Heavy Metal Concentrations in Wild Mussels Mytilus galloprovincialis (Lamarck, 1819) during 2001–2023 and Potential Risks for Consumers: A Study on the Romanian Black Sea Coast

Abstract

This study investigates the potential health risks associated with consuming mussels (Mytilus galloprovincialis Lamarck, 1819) from the Romanian Black Sea coast between 2001 and 2023. The research focuses on heavy metal (copper, cadmium, lead, nickel, and chromium) bioaccumulation in mussels and the associated human health hazards. While most metals fell within safe limits, lead and cadmium exceeded the maximum admissible concentrations set by the European Commission in a small percentage of samples (10% for cadmium, 14% for lead). To assess human health risks, we calculated dietary intake estimates and hazard quotients. These calculations suggested that current metal concentrations in the mussels are unlikely to cause adverse health effects at typical consumption levels. Although current metal concentrations seem safe based on estimated intake and hazard quotients, we emphasize the need for continuous monitoring of pollutants in seafood to ensure consumer safety. Future research should consider the cumulative effects of various contaminants and how individual factors like age and health conditions might influence risk. Public health protection requires continuous monitoring, comprehensive risk assessments, and transparent communication between scientists, policymakers, and the public to establish safe consumption guidelines.

1. Introduction

Anthropogenic activities are significantly elevating the presence of heavy metals (mercury, cadmium, lead, copper, etc.) within marine environments [1]. These pollutants originate from diverse sources, including industrial effluent discharge, agricultural runoff, or antifouling paints employed on ship hulls [2]. The principal environmental issue related to heavy metals is their persistence in the environment and their tendency to bioaccumulate in marine organisms [1]. Filter-feeding organisms and benthic invertebrates directly ingest these pollutants, and through biomagnification, their concentration progressively increases as they transfer through the food web, potentially reaching ecologically and toxicologically significant levels in commercially important fish species [2]). Emerging research suggests that heavy metal contamination disrupts various physiological processes in marine life, potentially impacting fish reproduction, larval development, and overall population health in species across trophic levels [3].

A study examining the effects of copper, cadmium, nickel, and their mixture on the reproductive health of the Mediterranean mussel (Mytilus galloprovincialis) revealed that exposure to these heavy metals, especially in combination, leads to significant alterations in protamine-like proteins, increased DNA damage, and changes in sperm chromatin structure. Heavy metal exposure disrupts gonadal tissue morphology, as shown by increased poly (ADP-ribose) polymerase (PARP) expression and sperm DNA fragmentation. This is coupled with elevated stress gene expression, suggesting a synergistic negative impact from combined metals compared to single exposures. The study concludes that heavy metals disrupt protein/DNA interactions, resulting in reproductive impairments in mussels, highlighting the need to understand the combined effects of multiple pollutants for accurate ecological and health risk assessments [4].

An innovative investigation explored the metabolic response of Mytilus galloprovincialis gonadal tissue to heavy metal (copper, cadmium, nickel, and their mixture) exposure. Significantly, the gonads exhibited a marked increase in metabolite abundance following copper and cadmium exposure. These findings suggest potential adverse effects on M. galloprovincialis reproductive health, particularly with cadmium. The observed alterations in metabolite profiles point toward a potential disruption of physiological processes. Certain metabolites, though potentially beneficial at low concentrations, may hinder sperm production and motility and cause tissue damage at high levels. The concerning rise in specific metabolites, especially cadmium, requires further studies. This study acknowledges the complexity of these interactions, with the possibility that some observed metabolite changes might represent a compensatory or protective response alongside potential detrimental effects. Discerning this intricate balance between positive and negative impacts is paramount to fully understanding the consequences of heavy metal exposure on M. galloprovincialis’ reproductive health [5].

Bioaccumulated heavy metals in seafood can pose a significant threat to human health upon consumption, with potential consequences ranging from neurological impairments to developmental abnormalities [3,6]. A comprehensive understanding of the sources, ecological consequences, and potential mitigation strategies for heavy metal pollution in marine ecosystems is paramount to ensure the health and sustainability of our oceans and safeguard human well-being.

In various marine regions, research has investigated the bioaccumulation of heavy metals in mussels (Mytilus galloprovincialis) to assess potential health risks, highlighting the importance of monitoring contaminant levels to ensure consumer safety [7,8]. Moreover, the utilization of mussels as bioindicators for monitoring heavy metal pollution has been investigated, alongside the evaluation of health risks associated with their consumption [9]. Initiatives have been undertaken to monitor and evaluate heavy metal concentrations in marine organisms, aiming to elucidate the extent of contamination and its potential ecological and health impacts [10].

The Black Sea ecosystem faces challenges from pollution originating from various sources such as industrial activities, urban sewage, and agricultural runoff [11,12]. Studies on water, sediments, and mussels, particularly in areas such as harbors, revealed significant heavy metal accumulation, highlighting a critical environmental concern that impacts various organisms and ecosystems within the region. [13]. Research has demonstrated that marine organisms, particularly macroalgae and mussels, function as bioaccumulators of heavy metals, rendering them valuable indicators of environmental contamination. Numerous studies have concentrated on evaluating the levels of heavy metals in sediments, coastal waters, and marine organisms, including mussels, along the Black Sea coastlines of countries such as Romania, Bulgaria, and Turkey [12,14,15,16]. These investigations aimed at quantifying the extent of pollution in the region and revealed that the accumulation of heavy metals in marine organisms (mussels and fish) could pose risks to both environmental and human health [11]. Mussels have been commonly used as bioindicators of heavy metal contamination in coastal waters. Studies have shown variations in heavy metal concentrations in mussels from different sampling areas along the Black Sea coast, providing valuable insights into environmental contamination and highlighting the need for biomonitoring to ensure consumer safety [17,18,19].

Studies have highlighted the potential dangers associated with toxic elements (cadmium, lead, arsenic, mercury) exposure through seafood consumption. The bioaccumulation of heavy metals in marine organisms (fish and shellfish) can lead to adverse health effects in humans [20]. While the levels of heavy metals in some seafood may be below allowable concentrations, long-term intake of contaminated seafood can still lead to heavy metal toxicity in humans. Health risk assessments revealed varying degrees of exposure to heavy metals in seafood over time, highlighting periods of potentially greater risk for consumers [21]. Studies have also emphasized the importance of monitoring heavy metal contents in marine environments and organisms to ensure seafood safety and protect consumer health [22]. Furthermore, the guidelines set by different countries and associations regarding heavy metals in seafood play a crucial role in ensuring consumer safety [23]. It is essential for consumers to be aware of the potential health risks associated with consuming contaminated seafood. Therefore, proper monitoring, assessment, and adherence to safety guidelines are necessary to mitigate the hazards posed by heavy metal contamination in seafood.

Many studies have utilized methods like the target hazard quotient (THQ) and hazard index (HI) to evaluate the health risks associated with heavy metal consumption through various food sources, including mussels [24]. These assessments involve estimating the probable daily intake (EDI) and hazard quotients (HQs) to determine human exposure risks [25,26]. The bioaccessibility of heavy metals (cadmium, lead, mercury) in benthic organisms collected from the Black Sea’s Turkish coast has been investigated to evaluate potential risks to human health [27]. Overall, while heavy metal concentrations in mollusks and fish may not exceed permissible limits set by legislation, potential human health risks persist, particularly with increased consumption frequency or portion sizes. The authors reiterate the necessity for continued biomonitoring of contaminants within the Black Sea biota to ensure the safety of seafood consumption and the ecological health of the marine ecosystem [27,28,29].

Long-term (2001–2023) data collected from a monitoring program along the Romanian Black Sea coast regarding heavy metal bioaccumulation in mussels have been partially published over time, addressing various scientific aspects, like exploring bioaccumulation patterns of specific heavy metals in mussels, evaluating their effectiveness as a bioindicator species for marine pollution, and comparing measured concentrations against maximum permissible levels for seafood consumption and environmental health [30,31]. Additionally, a portion of the data has been incorporated into reports of ecological status assessment of marine waters required under the implementation cycles (2012, 2018) of the Marine Strategy Framework Directive (MSFD) [32,33]. The MSFD reporting focuses on temporal trends of heavy metal levels in mussels, their impact on achieving good environmental status (GES) for the marine environment, and potential management measures to address any identified issues. This paper presents the first assessment of the potential human health risks associated with seafood consumption, based on the long-term monitoring data (2001–2023) on heavy metal accumulation in mussels (Mytilus galloprovincialis) from the Romanian Black Sea coast, using established methods like hazard quotients (THQ) and estimated daily intake (EDI).

2. Materials and Methods

2.1. Study Area

Romanian Black Sea sector experiences major impacts from the Danube’s discharge in the northern part and industrialized and urbanized areas in the southern part, together with a surge in tourism during the summer months. The Romanian coastline extends 244 km from Ukraine to Bulgaria, representing 6% of the total Black Sea coast. The northern sector is heavily influenced by freshwater inflows, particularly from the Danube River, which contributes 55% of the total freshwater input into the Black Sea. Other major rivers, including the Dniester, Dnieper, Don, and Bug, also significantly impact the region’s hydrography, chemistry, and biology. Key activities in the southern area of the coastline include wastewater treatment, beach nourishment, maritime transport, fishing, and oil and gas extraction. Wastewater treatment facilities, such as those at Rompetrol, Constanta Nord, Constanta Sud, Eforie, and Mangalia, have faced challenges in the past, though improvements were noted by 2018 due to upgrades to treatment processes, infrastructure enhancements, and capacity expansion. Maritime transport centers on the major ports of Constanta, Mangalia, and Midia, with Constanta being the largest Black Sea port. Offshore oil and gas exploration began in 1975, with significant production starting in 1987. Currently, the Black Sea contributes 8% of Romania’s oil and gas production, led by OMV Petrom, with future increases expected from the Neptun Deep field by 2027. Environmental pressures are substantial, mainly from shipping, coastal defense, and construction activities. Shipping exerts the highest impact, causing pollution and marine litter, while coastal defense and flood protection alter natural sediment dynamics. Dredging and offshore construction disrupt the seabed, leading to habitat loss and biodiversity changes. The long-term health of marine ecosystems necessitates a bridge between economic activities and environmental sustainability, achievable through the implementation of sustainable practices [34].

2.2. Sampling Procedure

During monitoring expeditions (2001–2023) on the R/V “Steaua de Mare 1”, 149 wild mussels (Mytilus galloprovincialis) samples were collected from the Romanian Black Sea sector. The marine monitoring network is represented by 45 stations, positioned on 13 transects along the Romanian coast, between Sulina and Vama Veche, on a bathimetric strip 10–100 m (Figure 1).

In the framework of the monitoring cruises, conducted usually in the warm seasons, along with seawater and sediments sampled for chemistry, contaminants, and biological parameters, mussels were collected using a biological dredge (Figure 2) along monitoring transects to be investigated for hazardous substances, including heavy metals. Bivalve mollusks (mussels) were collected across a range of Romanian Black Sea sites, including transitional and coastal areas influenced by anthropogenic activities (e.g., Danube River discharges, wastewater treatment plants, ports) and more pristine marine zones located farther from pollution sources, designated by the National Sanitary Veterinary and Food Safety Authority (ANSVSA) as suitable for live mussel harvesting [35]. This sampling strategy, spanning 2001–2023, captures the spatial variability of heavy metal concentrations in mussels potentially linked to environmental gradients.

A composite sample of mussels was obtained by selecting a minimum of 10–15 individuals with a shell length of 4–5 cm from each study site. This size range was chosen to ensure sufficient soft tissue for analysis and minimize variability due to differences in metal accumulation across various growth stages. Following sampling, mussels were rinsed with seawater on board the research vessel to remove any adhering debris, stored in freezer boxes, and transported to the laboratory for further analysis. The whole soft tissue of the mussels was carefully separated from the shells without damaging the visceral mass, which encompasses the digestive gland, gills, mantle, and other internal organs. The visceral mass is the primary site of bioaccumulation for heavy metals in mussels and represents the portion typically consumed by humans. Subsequently, samples were freeze-dried using a Labconco Freeze Dry System, homogenized with an electric grinder, and then analyzed for the presence of heavy metals [36].

2.3. Heavy Metals Analysis

For heavy metal analyses in mussels’ whole soft tissues, about 0.05–0.5 g of dry material was digested with 10 mL concentrated HNO₃ in sealed Teflon vessels on an electric hot plate at 120 °C. After mineralization, the solution was made up to 100 mL with deionized water (18.2 MΩ.cm, Millipore, Burlington, MA, USA). Heavy metal (HM) copper (Cu), cadmium (Cd), lead (Pb), nickel (Ni), and chromium (Cr) determinations were performed using the graphite furnace atomic absorbtion spectrometry (GF AAS) method on a High-Resolution Continuum Source AAS (HR-CS ContrAA 800 G equipment, Analytik Jena, Jena, Germany). Calibration was performed with working standards prepared from Merck stock solutions for each element in the following ranges: 0−50 µg/L (Cu), 0−10 µg/L (Cd), 0−25 µg/L (Pb), 0−50 µg/L (Ni), 0−50 µg/L (Cr). Each sample was measured in three replicates, and the average value was reported. The method detection limits for HMs were, depending on the element, between 0.001 and 0.01 µg/L. To ensure the accuracy of the analytical procedures, standard protocols were used [37]. Tissue concentrations are expressed as µg/g tissue wet weight (ww).

We minimized external contamination during heavy metal analysis by selecting high-purity materials throughout the analytical process, including glassware or reagents such as SUPRAPUR Nitric acid 65% and SUPRAPUR Deionized water, that are certified by the manufacturer to contain minimal trace metal levels. All glassware and equipment, including tubes and vials used for sample handling, were meticulously cleaned using established protocols known to remove potential metal contaminants (acid washes, rinsing with ultrapure water). Sample blank samples were analyzed alongside the mussel samples throughout the GF AAS analysis, consisting of all the reagents and materials used in the analysis but without the actual mussel tissue. This helps to identify and control any background contamination arising from the materials or analytical process. Our laboratory maintains strict environmental controls to minimize airborne contaminants and ensure the stability of our analyses. This includes temperature and humidity control, as well as regular maintenance and calibration of analytical instruments to uphold their performance.

2.4. Human Health Risk Assessment

In the present study, human health risk indices, including the estimated daily intake (EDI) and the estimated weekly intake (EWI), target hazard quotient (THQ), total hazard quotient (TTHQ), and carcinogenic risk index (CRI), were assessed for five heavy metals (Cu, Cd, Pb, Ni, Cr), to evaluate the probability of a health hazard associated with consuming mussels from the Romanian Black Sea. The analysis involved assessing the extent of mussel consumption and the corresponding intake of heavy metals. By integrating these factors with toxicological data, the study provided a comprehensive risk assessment of the potential adverse health effects associated with the ingestion of contaminated mussels.

2.4.1. Estimated Daily Intake (EDI) and Estimated Weekly Intake (EWI)

The average daily intake of heavy metals (mg/kg/day) must be considered when calculating risk exposure. The estimated daily intake (EDI) is calculated based on element levels and the amounts of mussels consumed. The following equation was used to calculate the estimated daily intake (EDI) of heavy metals [38,39]:

EDI (mg/kg/day) = (C_metal × FIR)/BW,

where EDI is the estimated daily intake of heavy metal (mg/kg/day), C_metal is the concentration of heavy metal in mussels (mg/kg, wet wt.), FIR (food ingestion rate) (kg/day) is the daily mean consumption of food item, and BW is the average body weight (30 kg for children and 70 kg for adults).

Information on the daily mean consumption of food item (FIR) was obtained from FAOSTAT [40]. Food supply quantity (kg/capita/year) for the category “Molluscs, Other” for Romania, period 2010–2021, varied between 0.08–0.50 kg/capita/year, respectively, 0.00022–0.00137 kg/day.

Estimated weekly intake (EWI) values were found by multiplying EDI values by 7.

2.4.2. Non-Carcinogenic Hazard: Target Hazard Quotient (THQ) and Hazardous Risk (Total Hazard Quotient) (TTHQ)

The non-carcinogenic risk related to the consumption of mollusks and their associated heavy metals was evaluated using the target hazard quotient (THQ) or hazard index (HI) [41], determined as the ratio of the calculated metal dosage (EDI mg/kg/day) to the reference dose (Rf. D. mg/kg/day):

THQ = EDI/Rf. D.,

where Rf. D. is the chronic oral reference dose (mg/kg/day), which refers to the estimated maximum permissible health risk associated with daily human consumption of metals in food items. The Rf. D. values for Cd, Cu, Co, Ni, and Cr are 0.0001, 0.04, 0.0003, 0.02, and 0.003 mg/kg/day, respectively (USEPA Regional Screening Levels (RSLs): [42]; The Risk Assessment Information System (RAIS): [43]). However, the Rf. D. value for Pb is not given.

If THQ > 1.0, the EDI of a particular metal exceeds the Rf. D., indicating that the metal is potentially hazardous. It is dependent on both metal levels and the amounts of mussels consumed.

The TTHQ estimates the cumulative risk associated with exposure to multiple heavy metals.

$$\mathrm{T}\mathrm{T}\mathrm{H}\mathrm{Q}={\sum }_{\mathrm{i}=1}^{\mathrm{n}}\mathrm{T}\mathrm{H}\mathrm{Q}\mathrm{i},$$

The total target hazard quotient (TTHQ), calculated by summing the hazard quotients (HQs) of individual metals, serves as an indicator of the potential health risk posed by exposure to multiple metals. When TTHQ exceeds 1, it suggests a potential chronic danger, indicating that the cumulative effect of the metals may lead to adverse health effects over time. Conversely, if TTHQ is less than 1, it implies that there is no potential health risk associated with the combined impacts of the metals.

This understanding is crucial in assessing the health implications of exposure to multiple metals simultaneously, as it provides insight into the complex interactions between different pollutants and their cumulative effects on human health. By evaluating TTHQ, researchers can better characterize the risks posed by exposure to metal mixtures and inform regulatory decisions aimed at mitigating potential health hazards associated with environmental contamination.

2.4.3. Carcinogenic Risk Index (CRI)

The CRI is one metric to measure the carcinogenic risk. The equation below represents CRI in terms of:

CRI = EDI × CSF,

where CSF (cancer slope factor) (mg/kg/day)⁻¹ establishes the risk associated with a lifetime average contaminant dose. CSF value is given for Pb, and this value is 0.0085 (mg/kg/day)⁻¹, according to USEPA Regional Screening Levels (RSLs): [42] and the Risk Assessment Information System (RAIS): [43].

If the CRI is less than 10⁻⁶, it is deemed inconsequential; if the CRI is between 10⁻⁶ and 10⁻⁴, it is acceptable or bearable; and if the CRI is greater than 10⁻⁴, it is deemed significant.

The data were processed using MS Excel 365, Statistica (TIBCO Software Version 14.0.1.25) [44], and Ocean Data View (ODV) version 5.1.7 [45]. Distribution maps in ODV were generated through weighted-average gridding interpolation.

3. Results

3.1. Heavy Metal Variability in Black Sea Mussels

Table 1. Heavy metals concentrations variability and distribution in mussels (Mytilus galloprovincialis) from the Romanian Black Sea coast investigated during 2001–2023.

Element	N	Mean	Coef.Var.	Median	Min	Max	75th Percentile	Skewness	Kurtosis
Cu (µg/g ww)	149	2.617	73.818	2.259	0.100	10.770	3.344	1.745	4.111
Cd (µg/g ww)	149	0.479	130.625	0.303	0.006	4.690	0.4720	3.638	17.260
Pb (µg/g ww)	149	0.790	213.443	0.160	0.001	11.020	0.741	4.006	18.389
Ni (µg/g ww)	125 *	1.223	97.491	0.950	0.118	8.120	1.496	3.154	13.071
Cr (µg/g ww)	106 *	0.859	125.395	0.467	0.002	6.076	1.074	2.578	7.619

* Nickel and chromium were analyzed in samples starting with 2003 and 2004, respectively; this explains fewer samples number.

and Figure 3 show the variability and distribution of heavy metal concentrations in mussels (Mytilus galloprovincialis) from the Romanian Black Sea coast investigated during 2001–2023. Overall, the analysis reveals significant differences. Lead (Pb) shows the highest variability and a potential for high concentrations, while copper (Cu) has the least variability. Cadmium (Cd) shares similarities with Pb in terms of variability and potential for high concentrations, while nickel (Ni) and chromium (Cr) exhibit a wider range of concentrations with fewer extreme outliers.

Pb shows the highest coefficient of variation (CoV) (213.443), indicating the most significant variability in its concentration among the five metals. Cu (73.818) has the least variable concentration, implying a more consistent level of Cu found in the mussels. Cd (130.625), Ni (97.491), and Cr (125.395) exhibit moderate CoV values, suggesting some variability in their concentrations but not as significant as Pb.

The skewness and kurtosis coefficients are measures of the shape of the distribution. Table 1 shows that the distributions of all five metals (Cu, Cd, Pb, Ni, Cr) show positive skewness values. This indicates that the distributions are skewed to the right, with a longer tail extending towards the higher concentrations. In simpler terms, there are fewer mussels with concentrations close to the minimum values and a larger number with concentrations closer to the mean, with a few outliers with higher concentrations than the average. A skewed distribution to the right is visually evident in the histograms, with most of the bars being on the left side of the graph, representing the larger number of mussels with lower concentrations, and the tails extending towards higher concentrations being represented by a smaller number of bars on the right side (Figure 3).

The kurtosis coefficients for all five heavy metals are greater than 3, indicating that the distributions are more peaked than a normal distribution (Table 1). This suggests that there is a central tendency in the data, with most of the samples having concentrations of heavy metals that are close to the mean, but there are also a few mussels with extreme concentrations (outliers). Pb (18.389) and Cd (17.260) have the highest kurtosis values, indicating a sharper peak around the central tendency compared to the other metals. This suggests a more pronounced clustering of mussels with concentrations close to the mean, with a steeper decline towards the tails (both lower and higher ends). A sharper peak of the histogram suggests a more pronounced clustering of data points near the mean, as seen with higher kurtosis values for Pb and Cd. The presence of outliers with high concentrations is shown by bars on the far right of the histogram, even if they are a smaller number compared to the mussels with closer-to-mean concentrations (Figure 3).

Overall, lead (Pb) exhibits the highest variability and the most prominent positive skewness, suggesting a larger number of mussels with Pb concentrations closer to the mean, but also a significant presence of mussels with higher Pb concentrations. Copper (Cu) shows the least variability and a moderate positive skewness. This suggests a more consistent level of Cu in the mussels, with a smaller number of mussels having extreme Cu concentrations. While exhibiting moderate variability, Cd has the second-highest positive skewness and the second-highest kurtosis. This suggests a similar pattern to Pb, with a larger number of mussels with Cd concentrations closer to the mean but also a significant presence of mussels with higher Cd concentrations. Ni and Cr show moderate variability and positive skewness but with lower kurtosis values compared to Pb and Cd. This suggests a less pronounced clustering around the mean and a more gradual decline towards the tails, indicating a wider range of Ni and Cr concentrations in the mussels but with fewer extreme outliers compared to Pb and Cd. It is important to mention that, overall, these extreme values of heavy metals concentrations represented less than 10–15% of total mussels samples investigated during 2001–2023 (Figure 3).

The variability in heavy metal levels in mussels, as sessile and filter-feeding organisms, is significantly influenced by their proximity to pollution sources, such as industrial and urban areas. The ability of mussels to integrate and reflect the contamination over time makes them valuable for monitoring temporal and spatial trends of heavy metal pollution. Mussels collected from areas closer to various pollution sources exhibited higher concentrations of heavy metals compared to those from more pristine areas. The spatial distribution maps generally show higher bioaccumulation levels of heavy metals near major ports, such as Port Midia, Port Constanta, and Port Mangalia, due to industrial activities, shipping, wastewater discharges, and urban runoff. The areas in front of the Danube Delta also show moderate levels of heavy metals, reflecting the influence of riverine inputs carrying pollutants from upstream. Lower concentrations of heavy metals in mussels offshore and in more open waters suggest the dilution and dispersion effects away from the coastal sources of pollution. (Figure 4). The spatial distribution maps highlight that all investigated metals have similar high concentration areas near major ports and the Danube Delta, pointing to common sources such as industrial activities and riverine inputs. However, there are similarities and differences in their distribution patterns, which can provide insights into their sources and environmental behaviors. Copper (Cu) shows relatively uniform distribution with some localized higher concentrations, while cadmium (Cd) exhibits more significant variability with higher concentrations in specific areas. Chromium (Cr) and nickel (Ni) display similar patterns, with higher concentrations near urban and industrial zones, while lead (Pb) has the highest variability and potential for high concentrations. These were confirmed by the skewed distribution and high kurtosis for Cd, Pb, and Ni, suggesting a central tendency with occasional extreme values (Table 1).

The variability of annual mean values of heavy metals concentrations in mussels over a 23-year period is depicted in Figure 5. Overall, while there are notable fluctuations over time, all metals showing significant outliers and extremes in certain years, no significant increasing trend in heavy metal concentrations is observed over time, which suggests improved pollution control measures in the last period. On the contrary, there has been a general trend towards stabilization and lower mean values, particularly for lead, in recent years. However, the presence of outliers and extreme values measured, usually in mussels from hotspot areas, underscores the need for ongoing environmental monitoring and pollution mitigation efforts to ensure long-term safety and sustainability.

3.2. Compliance with European Regulations

The widespread consumption of mussels necessitates rigorous monitoring of heavy metal bioaccumulation to mitigate potential human health risks. This practice serves a dual purpose, safeguarding both ecosystem health and public well-being. Ensuring that mussels harvested for human consumption comply with established safety standards is crucial in mitigating these risks. Consumption of contaminated mussels can lead to adverse health effects, including heavy metal poisoning, which underscores the necessity of stringent regulatory measures and continuous surveillance of mussel populations in contaminated areas.

In the present study, Cd and Pb values measured in mussels during 2001–2023 were compared to the values permitted by European Commission Regulation (EC) 2023/915 [46] for consumed seafood. Exceeding the maximum admissible concentration (MAC) (1.50 µg/g ww Pb; 1 µg/g ww Cd) was more frequent for lead (Pb) (14%) than cadmium (Cd) (10%) (Figure 6). Most contaminated samples originated from coastal areas impacted by human activities, including Danube River discharge, wastewater outflows, and harbor operations (Figure 3). Cadmium (Cd) displayed higher mean concentrations in 2001, 2007, 2014, 2016, and 2019, while lead (Pb) had significant peaks in 2001, 2005, 2009, and 2011 (Figure 5). These results emphasize the need for ongoing monitoring of contaminants in marine life to ensure safe seafood.

3.3. Human Health Risk Assessment

This study represents a comprehensive assessment of health risks from consuming mussels in Romania, based on the long-term monitoring data (2001–2023). Estimated daily intake (mg/kg/day) (EDI), estimated weekly intake (mg/kg/week) (EWI), target hazard quotient (THQ), total hazard quotient (TTHQ), and carcinogenic risk index (CRI) were calculated based on annual mean values of heavy metals. The detailed results for each year can be found in Tables S1–S3 of the Supplementary Material. To facilitate easier reading and comparison,

Table 2. The EDIs and EWIs for heavy metals, THQs, and TTHQ for Cu, Cd, Cr, and Ni, and carcinogenic risk index (CRIs) for Pb in two groups consuming mussels (Mytilus galloprovincialis) from the Romanian sector of the Black Sea (2001–2023).

Metal	Period	EDIs		EWIs		THQs		CRIs
		Children	Adults	Children	Adults	Children	Adults	Children	Adults
Copper (Cu)	2001–2010	2.16 × 10−5 ± 9.70 × 10−6	9.27 × 10−6 ± 4.15 × 10−6	1.51 × 10−4 ± 6.79 × 10−5	6.49 × 10−5 ± 2.91 × 10−5	5.41 × 10−4 ± 2.42 × 10−4	2.32 × 10−4 ± 1.04 × 10−4
	2011–2020	8.43 × 10−5 ± 3.19 × 10−5	3.61 × 10−5 ± 1.36 × 10−5	5.90 × 10−4 ± 2.23 × 10−4	2.53 × 10−4 ± 9.57 × 10−5	2.11 × 10−3 ± 7.98 × 10−4	9.03 × 10−4 ± 3.42 × 10−4
	2021–2023	4.06 × 10−5 ± 2.17 × 10−6	1.74 × 10−5 ± 9.31 × 10−7	2.84 × 10−4 ± 1.52 × 10−5	1.22 × 10−4 ± 6.51 × 10−6	1.02 × 10−3 ± 5.40 × 10−5	4.35 × 10−4 ± 2.32 × 10−5
Cadmium (Cd)	2001–2010	4.07 × 10−6 ± 5.39 × 10−6	1.75 × 10−6 ± 2.31 × 10−6	2.85 × 10−5 ± 3.78 × 10−5	1.22 × 10−5 ± 1.61 × 10−5	4.07 × 10−2 ± 5.39 × 10−2	1.75 × 10−2 ± 2.31 × 10−2
	2011–2020	1.56 × 10−5 ± 9.98 × 10−6	6.71 × 10−6 ± 4.27 × 10−6	1.10× 10−4 ± 6.98 × 10−5	4.69 × 10−5 ± 2.99 × 10−5	1.56 × 10−1 ± 9.98 × 10−2	6.71 × 10−2 ± 4.27 × 10−2
	2021–2023	1.48 × 10−5 ± 1.98 × 10−6	6.36 × 10−6 ± 8.51 × 10−7	1.04 × 10−4 ± 1.39 × 10−5	4.45 × 10−5 ± 5.96 × 10−6	1.48 × 10−1 ± 1.98 × 10−2	6.36 × 10−2 ± 8.51 × 10−3
Chromium (Cr)	2004–2010	9.50 × 10−6 ± 1.05 × 10−5	4.07 × 10−6 ± 4.52 × 10−6	6.65 × 10−5 ± 7.39 × 10−5	2.85 × 10−5 ± 3.16 × 10−5	3.17 × 10−3 ± 3.52 × 10−3	1.36 × 10−3 ± 1.51 × 10−3
	2011–2020	2.12 × 10−5 ± 2.61 × 10−5	9.07 × 10−6 ± 1.11 × 10−5	1.48 × 10−4 ± 1.82 × 10−4	6.35 × 10−5 ± 7.83 × 10−5	7.05 × 10−3 ± 8.70 × 10−3	3.02 × 10−3 ± 3.72 × 10−3
	2021–2023	3.97 × 10−5 ± 4.03 × 10−5	1.70 × 10−5 ± 1.73 × 10−5	2.78 × 10−4 ± 2.82 × 10−4	1.19 × 10−4 ± 1.21 × 10−4	1.32 × 10−2 ± 1.34 × 10−2	5.67 × 10−3 ± 5.77 × 10−3
Nickel (Ni)	2003–2010	1.09 × 10−5 ± 8.37 × 10−6	4.67 × 10−6 ± 3.58 × 10−6	7.64 × 10−5 ± 5.86 × 10−5	3.27 × 10−5 ± 2.51 × 10−5	5.45 × 10−4 ± 4.18 × 10−4	2.34 × 10−4 ± 1.79 × 10−4
	2011–2020	3.49 × 10−5 ± 1.89 × 10−5	1.50 × 10−5 ± 8.12 × 10−6	2.45 × 10−4 ± 1.32 × 10−4	1.05 × 10−4 ± 5.68 × 10−5	1.75 × 10−3 ± 9.47 × 10−4	7.49 × 10−4 ± 4.06 × 10−4
	2021–2023	2.56 × 10−5 ± 1.48 × 10−5	1.10× 10−5 ± 6.36 × 10−6	1.80 × 10−4 ± 1.04 × 10−4	7.69 × 10−5 ± 4.45 × 10−5	1.28 × 10−3 ± 7.42 × 10−4	5.50 × 10−4 ± 3.18 × 10−4
Lead (Pb)	2001–2010	9.05 × 10−6 ± 1.14 × 10−5	3.88 × 10−6 ± 4.89 × 10−6	6.33 × 10−5 ± 8.02 × 10−5	2.71 × 10−5 ± 3.42 × 10−5			7.69 × 10−8 ± 9.71 × 10−8	3.30 × 10−8 ± 4.16 × 10−8
	2011–2020	3.30 × 10−6 ± 4.02 × 10−6	1.41 × 10−6 ± 1.72 × 10−6	2.31 × 10−5 ± 2.82 × 10−5	9.90 × 10−6 ± 1.20 × 10−5			2.81 × 10−8 ± 3.42 × 10−8	1.20 × 10−8 ± 1.46 × 10−8
	2021–2023	2.49 × 10−5 ± 1.43 × 10−5	1.07 × 10−5 ± 6.13 × 10−6	1.75 × 10−4 ± 1.01 × 10−4	7.48 × 10−5 ± 4.29 × 10−5			2.12 × 10−7 ± 1.21 × 10−7	9.10× 10−8 ± 5.21 × 10−8
TTHQ	2001–2010					4.28 × 10−2± 5.40 × 10−2	1.87 × 10−2 ± 2.28 × 10−2
	2011–2020					1.66 × 10−1 ± 1.05 × 10−1	7.14 × 10−2 ± 4.52 × 10−2
	2021–2023					1.68 × 10−1 ± 7.17 × 10−3	7.18 × 10−2 ± 3.07 × 10−3

EDI: Estimated daily intake (mg/kg/day); EWI: Estimated weekly intake (mg/kg/week); THQ: Target hazard quotient; TTHQ: Total hazard quotient; CRI: Carcinogenic risk index.

presents the data for EDI, EWI, THQ, TTHQ, and CRI (average values ± standard deviation) grouped into three periods: 2001–2010, 2011–2020, and 2021–2023.

Our findings on the estimated daily intake (EDI) and weekly intake (EWI) of heavy metals for two distinct groups consuming mussels (Mytilus galloprovincialis) show that the EDI rates for copper (Cu), cadmium (Cd), chromium (Cr), nickel (Ni), and lead (Pb) were consistently below the chronic oral reference dose (Rf. D.) for both children and adults, suggesting safe consumption levels.

The calculated exposure values (EDIs) were compared and found below the health-based guidance values (HBGV) provided by the European Food Safety Authority (EFSA) [39]. For lead (Pb), the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) both established an HBGV of 6.3 × 10⁻⁴ mg/kg/day ([47]). Similarly, EFSA’s CONTAM Panel recently set a tolerable daily intake (TDI) of 1.3 × 10⁻² mg/kg/day for nickel (Ni) after a request from the European Commission to update its previous assessment considering new data [48]. Finally, for cadmium (Cd), the CONTAM Panel recommends a tolerable weekly intake (TWI) of 2.5 × 10⁻³ mg/kg/week, which is equivalent to 3.6 × 10⁻⁴ mg/kg/day [49].

Table 2 also provides the target hazard quotients (THQs) and total hazard quotient (TTHQ) values for copper (Cu), cadmium (Cd), chromium (Cr), and nickel (Ni). If the THQ exceeds 1.0, it indicates that the estimated daily intake (EDI) of a specific metal surpasses the chronic oral reference dose (Rf. D.), suggesting potential health hazards.

The THQ is influenced by both metal concentrations and the quantities of mussel consumption. Metal accumulation in mussels along the Romanian Black Sea coast stems from a confluence of human activities and natural processes. These factors include industrial and urban wastewater discharge, riverine input, and atmospheric deposition. These sources introduce a variety of metals into the marine environment, leading to elevated concentrations in specific areas. Based on the findings from our current study, the calculated target hazard quotients (THQs) for both children and adults during the period 2001–2023 indicate that the estimated values remain below levels of concern.

The total hazard quotient (TTHQ) provides an assessment of the cumulative risk associated with exposure to multiple heavy metals. If:

TTHQ > 1: Indicates potential chronic danger, suggesting that the combined effects of metals exceed safe levels;

TTHQ < 1: Implies no potential health risk, as the cumulative impact remains below critical thresholds.

In our study covering the period from 2001 to 2023, the TTHQ values were consistently lower than 1, indicating a negligible risk of adverse health effects from mussel consumption. However, based on estimated TTHQ values, children are at a higher risk of exposure compared to adults due to lower body weight (30 kg) compared to adults (70 kg), considering a similar consumption rate. An upward trend in annual TTHQ values was observed, particularly between 2014 and 2019, for both children and adults (Figure 7). This increasing trend suggests a potential for future health risks if heavy metal levels and consumption rates continue to rise. Although Figure 4 does not reveal significant increases in heavy metal concentrations over time, some elements displayed some peaks in their annual mean values in this interval. Furthermore, the higher TTHQ values can also be attributed to increased mussel consumption rates, according to available data from FAOSTAT [40]. Daily consumption of mussels in Romania ranged from 0.000219 to 0.001370 kg/day during the study period, as follows: available statistical data starting with the year 2010—food ingestion rate (FIR) of 0.000219 kg/day (this value was also used for quotients calculations for the previous period 2001–2009); FIR values gradually increased up to a maximum of 0.001370 kg/day in 2017–2018, followed by a slow decrease up to 0.001288 kg/day in 2021 (value that we used in calculations for 2022 and 2023, since statistical data are not available yet for the last years) (Figure 8).

In the case of lead (Pb), our findings reveal that the calculated carcinogenic risk index (CRI) associated with Pb exposure is negligible, being less than 10⁻⁶.

4. Discussion

Overall, our results evinced a considerable variability in the concentrations of heavy metals in mussels from the Romanian Black Sea coast during 2001–2023. The concentrations of heavy metals are skewed to the right, with a few mussels having higher concentrations than the average. The distributions are also more peaked than a normal distribution, suggesting that there is a central tendency in the data, but there are also a few outliers. Our analysis revealed that cadmium (Cd) and lead (Pb) exceeded MACs set by EC Regulation 2023/915 for seafood [46] in 10–14% of mussels. These contaminated samples were primarily found in coastal areas influenced by the Danube River, wastewater discharges, and harbor operations.

Several studies have documented the potential influence of seasonality on heavy metal accumulation in mussels, highlighting the importance of considering seasonal factors for a comprehensive understanding of metal accumulation patterns [7,50]. Azizi et al. (2021) [51] observed higher bioaccumulation in Mytilus galloprovincialis during winter, attributing this to fluctuations in heavy metal contents depending on the animals’ weight variation during the reproductive period. Similarly, Bat et al. (2012) [19] noted seasonal variations in heavy metal concentrations in bivalves, with the highest levels typically observed in late winter. Nardi et al. (2017) [52] explored the indirect effects of climate changes on cadmium bioavailability and biological effects in these mussels, emphasizing the influence of temperature and pH variations on cadmium accumulation. Overall, these studies collectively demonstrate that seasonal variations, particularly related to the reproductive cycle and physiological processes, significantly influence the bioaccumulation processes, emphasizing the importance of considering seasonality in assessing metal contamination levels in these organisms.

One of the limitations of our study is that most data consist of samples collected during the warm seasons, coinciding with routine monitoring cruises typically conducted in the summer months. While interesting, investigating potential seasonal variations in metal accumulation falls outside the scope of this study due to the limited data for other seasons. As future research directions, we recognize the value of exploring seasonal fluctuations in heavy metal accumulation. Building on the current dataset, future studies incorporating a broader sampling timeframe across different seasons would provide valuable insights into the seasonal influence on metal concentrations in mussels.

Our results strengthen the case for sustained monitoring of both abiotic and biotic components within the Black Sea marine environment to safeguard the safety of seafood consumption. Areas exhibiting heightened anthropogenic influence, including river deltas, industrial zones, wastewater outfalls, and shipping lanes, have an increased susceptibility to contaminant accumulation.

This study offers an initial assessment of heavy metal impacts in mussels. While the evaluated metals (Cu, Cd, Pb, Ni, Cr) show no apparent consumer risk based on calculated hazard quotients, these conclusions are limited to this specific set. Previous research has documented the presence of additional pollutants in Romanian Black Sea mussels, such as polycyclic aromatic hydrocarbons (PAHs) and persistent organic pollutants (POPs) [30,31,33,53]. These initial findings on heavy metals establish a groundwork for future investigations. A more comprehensive evaluation of human health risks requires considering the combined effects of hazardous chemicals, including persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs), alongside heavy metals (HMs) in mollusks. Understanding the long-term health implications is critical. Complex interactions and potential synergistic effects between these pollutants can magnify their combined toxicity, posing significant risks. Future research should broaden its scope to encompass not only the cumulative effects of HMs, POPs, and PAHs in mollusks but also their presence in fish, a more widely consumed seafood in Romania. This comprehensive approach will provide a clearer picture of the human health risks associated with these contaminants.

Our findings concur with studies on mussels from the Turkish Black Sea coast. Both sets of data suggest safe human consumption based on estimated daily intake (EDI) and target hazard quotient (THQ) values below 1, indicating negligible health risks [12,28,29,41]. Analysis of heavy metal concentrations in mussels from the Turkish coast also revealed significant variations between sampling locations. The concentrations (mg metal kg⁻¹ wet wt.) of metals ranged from 18 to 35 for Fe, 8 to 27 for Zn, 2.8 to 4.5 for Mn, 0.5 to 1.8 for Cu, 0.06 to 0.31 for Pb, 0.04 to 0.10 for Cd, and 0.03to 0.07 for Hg. Despite these variations, estimated daily intake (EDI) and total hazard index (THI) values fell below established safety thresholds, suggesting no chronic health risks associated with consuming mussels from this region. The authors also reviewed previous studies on mussels in Black Sea countries, consolidating data on heavy metal levels in mussels across the region. While the study found no health concerns associated with consuming mussels, data on typical consumption patterns are rather limited. To ensure safe consumption practices, a moderate intake of mussels, around one serving per week from clean coastal areas, was recommended [12].

Similar studies on the presence of heavy metals in mussels and the associated human health risks were conducted in the Black Sea region. One study investigated the levels and potential human health risks associated with metals in wild and farmed mussels from Bulgaria. The concentrations of Cd, Cr, Cu, Fe, Ni, Pb, and Zn were below the maximum permissible limits. Various hazard indices were also employed to assess potential health risks, and the results revealed safe consumption levels [54].

The are many studies that collectively contributed to understanding the distribution of heavy metals in mussels and the associated risks to human health in different marine regions. A comprehensive analysis of Cu, Zn, Pb, and Cd in marine mussels (Perna viridis, Meretrix sp., and Anadara granosa) from the Madura Strait in Indonesia, assessing bioaccumulation factors and health risk through target hazard quotients was conducted. Their findings indicated varying concentrations of heavy metals but with no immediate health risks for consumers. However, given the potential for long-term health effects and the presence of other contaminants, further investigations were recommended by the authors to comprehensively assess the safety of mussel consumption [55].

Investigations on mussels (Mytilus galloprovincialis) collected from the Mediterranean Sea revealed no immediate public health risks associated with moderate consumption. Concentrations of Cd, Cr, Cu, Hg, Pb, Zn, and As fell within permissible limits set by the European Commission and FAO. However, continued monitoring of heavy metal levels in mussels from this region is considered crucial to safeguard consumer health over time [7].

A literature review highlighted the health risks associated with the consumption of Mediterranean mussels (Mytilus galloprovincialis) due to heavy metal contamination. The study underscored the importance of measuring metal concentrations to evaluate potential adverse effects on human health. Data analysis revealed significant variations in the levels of Cd, Pb, Hg, and As in M. galloprovincialis from regions such as the Mediterranean, Adriatic, and Black Sea, with the general order of concentration being As > Pb > Cd > Hg. While the levels of these toxic metals in mussels typically remained below European regulatory limits, exceptions were noted in polluted areas like lagoons and harbors. Since M. galloprovincialis is a filter feeder, the metal concentrations in their soft tissue reflect marine ecosystem contamination. Consequently, monitoring the levels of pollutants in mussels is crucial due to the ongoing concerns regarding their consumption as seafood [23].

These studies emphasize the critical significance of monitoring heavy metal concentrations in mussels across diverse geographic regions to uphold human health standards and guide regulatory interventions. This research provides crucial insights into the bioaccumulation patterns of contaminants, health risk assessments through hazard quotients, and the importance of monitoring hazardous substances levels for human health protection. By synthesizing data from various studies, we can gain a comprehensive understanding of the distribution and impact of heavy metals pollution. This knowledge can guide policies aimed at ensuring seafood safety for consumers and preserving marine ecosystems.

Overall, our findings suggest that while the current level of heavy metals in mussels from the Romanian Black Sea poses a low risk for human health, there are reasons to be cautious. Children are more vulnerable than adults due to their lower body weight (30 kg). Additionally, the increasing levels of hazard quotients observed in recent periods may be attributed to higher concentrations of heavy metals, together with increased rates of mussel consumption. Continuous monitoring is necessary to ensure further consumer safety. The combination of spatial distribution and temporal trend data provides a comprehensive understanding of heavy metal pollution on the Romanian Black Sea coast. These insights are crucial for informing regulatory policies, public health guidelines, and future research on marine pollution.

The cumulative impact of heavy metals and other hazardous substances, such as organic pollutants, in mollusks and fish warrants further studies. Prolonged excessive consumption can pose health risks, and individual factors like age, weight, and existing health conditions should be considered. Transparent communication between scientists, policymakers, and the public is essential. Providing clear, science-based guidelines on safe consumption levels and emphasizing that occasional seafood consumption within these limits is safe is crucial.

Continuous monitoring of heavy metals and other pollutants in mussels and fish is essential. Regular sampling and analysis enable us to track fluctuations and trends over time. Collaboration between environmental agencies, research institutions, and seafood industry stakeholders is necessary to establish monitoring programs. These programs can identify potential contamination sources and assess the overall health of marine ecosystems.

5. Conclusions

The analysis of distribution maps and temporal trends of heavy metals in mussels, coupled with hazard quotient calculations, reveals a nuanced picture of heavy metal pollution along the Romanian Black Sea coast, evincing that there are periods and areas of concern.

This long-term study reveals significant spatial variations in the levels of heavy metals in mussels (Mytilus galloprovincialis) across the Romanian Black Sea coast. Areas with elevated pollutant levels, such as those influenced by the Danube, harbors, wastewater discharges, increased maritime traffic, and offshore oil and gas platforms, can significantly contribute to the bioaccumulation of heavy metals. This issue raises significant concerns about food safety and poses potential risks to human health. Our findings indicate that heavy metal exposure from consuming mussels does not present significant health risks to humans based on evaluations of estimated daily intake (EDI) and target hazard quotients (THQs). Despite these results, it is important to consider the potential cumulative effects of heavy metals, along with other contaminants present in mollusks. Such interactions may have synergistic or additive effects that are not captured by individual assessments. Therefore, further research should concentrate on the combined impact of multiple pollutants to offer a broader understanding of the associated risks to human health. Holistic risk assessment is key to safeguarding food safety and public health in polluted environments.

While seafood offers many health benefits, consuming excessive amounts over time can pose health risks. To ensure safe consumption, clear guidelines are needed. Transparent communication between scientists, policymakers, and the public is crucial for establishing these evidence-based recommendations. It is important to emphasize that occasional consumption of seafood within recommended limits is generally safe. Clear communication empowers the public to make informed dietary choices. This knowledge helps them mitigate potential health risks associated with long-term exposure to contaminants in food.

Our findings highlight the need for continuous monitoring of the marine environment, especially near the Danube discharge and other pollution hotspots. This ongoing surveillance, crucial for ensuring seafood safety, should prioritize areas like river mouths, industrial zones, wastewater outflows, shipping lanes, and coastal regions with past pollution or high human activity.

Marine mussels serve as reliable sentinel species in biomonitoring programs, providing crucial insights into the ecological health of marine ecosystems. The significant variability in heavy metal accumulation influenced by environmental factors such as proximity to pollution sources emphasizes the need for targeted monitoring and mitigation strategies. Furthermore, the risks posed to human consumers highlight the importance of stringent safety measures and public education to prevent health hazards associated with heavy metal contamination in seafood. This comprehensive analysis provides insights crucial for Black Sea environmental management and pollution mitigation. It emphasizes the urgent need for proactive measures to safeguard both the marine environment and human health. The findings highlight the criticality of targeted actions and policies to address and reduce pollution levels, ultimately promoting a healthier and more sustainable Black Sea ecosystem.